CN108841825A - Specificity MicroRNA sequence relevant to potassium-channel Eag1 expression and its application - Google Patents

Specificity MicroRNA sequence relevant to potassium-channel Eag1 expression and its application Download PDF

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CN108841825A
CN108841825A CN201810604285.8A CN201810604285A CN108841825A CN 108841825 A CN108841825 A CN 108841825A CN 201810604285 A CN201810604285 A CN 201810604285A CN 108841825 A CN108841825 A CN 108841825A
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sequence
eag1
microrna
expression
application
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李皓
李占东
朱可彤
李晓红
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Jilin Teachers Institute of Engineering and Technology
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Jilin Teachers Institute of Engineering and Technology
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Abstract

The present invention relates to a kind of specificity microRNA sequence relevant to potassium-channel Eag1 expression and its applications, belong to field of biological detection.The microRNA sequence such as SEQ ID NO：Nucleotide sequence shown in 1 has at least 70% sequence identity with it, preferably with the nucleotide sequence of 80%, 85%, 90%, 93%, 95%, 97%, 98%, 99% sequence identity.

Description

Specificity MicroRNA sequence relevant to potassium-channel Eag1 expression and its application
Technical field
The present invention relates to a kind of specificity microRNA sequence relevant to potassium-channel Eag1 expression and its applications, belong to In field of biological detection.
Background technique
MicroRNAs (i.e. miRNAs) is a hot spot in molecular biology research field in recent years, its maturity state It is a kind of small single strand RNA molecule for being about the skilful acid of 19-23 core, has in evolution well-conserved.The major function of miRNA is Adjust in organism gene related with body growth, development, disease generating process expression.Since participation regulates and controls nematode Since the lin-4 and let-7 of timing development are found, miRNA is selected in Science magazine in 2002 and 2003 two degrees respectively Annual ten big technological breakthroughs.Prediction miRNAs in 2005 can at least regulate and control 5300 human genes, i.e., and the 30% of all genes.With Research go deep into, more and more miRNAs are constantly found.MiRNA under spotlight gradually gets rid of DNA radiance Cover.In recent years, the relationship of miRNA and disease has become the focus and emphasis of research, it has been found that miRNA is adjusted by negative The expression for controlling gene and the morbidity of a variety of human diseases such as tumour, diabetes are highly relevant.
Research has confirmed that there are several hundred kinds of miRNAs in serum/tissue, transport a little microRNA s properties stabilizations, content It enriches, be easy to quantitative detection, and there are significant disease specifics.The technology of existing maturation, including qualitative and quantitative miRNA The technology of molecule shows using miRNAs the method for Molecular biomarkers than traditional differential protein molecule labelling method Will be more efficient, frontier has been opened up for biomarker.
Eag1 potassium-channel category voltage-gated potassium channel contains the single hole channel of 6 transmembrane regions (S1-S6), by going It polarizes and activates.Its distribution is extremely limited under normal circumstances, single-minded to be distributed in brain, only has in sarcoblast and placenta a small amount of Expression, but follow-up study finds that it has high expression in kinds of tumors.It is now recognized that tumor suppressor gene VHL is expressed in tumor tissues Reduction can activate the channel Eag1, enhance the stability of HIF-1 α, and the studies above result prompts the channel Eag1 may be in tumour cell Its main function in anoxic impression and the regulation of HIF-1 α.Chinese patent CN201720900420 discloses a kind of detection Eag1 egg White kit carries out related quantitative detection by ELISA principle.But Eag1 albumen is in blood of human body sample content pole It is low, be difficult qualitatively or quantitatively to be detected, and tumor tissue biopsy process is cumbersome, patient adaptability is poor, thus find with The relevant miRNA of Eag1 protein expression is a kind of relatively good method to solve the above problems.
Summary of the invention
The first aspect of the present invention is to provide a kind of specificity microRNA relevant to potassium-channel Eag1 expression, Sequence such as SEQ ID NO：Nucleotide sequence shown in 1 or with its have at least 70% sequence identity, preferably have 80%, 85%, the nucleotide sequence of 90%, 93%, 95%, 97%, 98%, 99% sequence identity.
The second aspect of the present invention provides application of the microRNA sequence in preparation detection Eag1 protein reagent box.
The third aspect of the present invention provides application of the microRNA sequence in preparation lesion detection kit.
In one embodiment, the tumour is selected from cervical carcinoma, oophoroma or lung cancer.
It is to provide a kind of kit for detecting the microRNA sequence in fourth aspect present invention, contains described The amplimer of microRNA sequence and the necessary reagent for extracting RNA.
In one embodiment, the sequence of the amplimer such as SEQ ID NO：Shown in 2-4.
In another embodiment, the kit also includes enzyme and reagent used in PCR reaction.
MiRNAs is a kind of new biomarkers, is different from traditional biological marker, not only stable, minimally invasive, be easy to examine Survey, and quantitative accurate, the sensibility and specificity of medical diagnosis on disease will be greatly improved, such microRNA biomarker at Function exploitation will start completely new situation for the early diagnosis of cervical carcinoma, offer reference for the development of other diseases biomarker.This Its expression quantity of miRNA that invention provides is positively correlated with Eag1 protein expression, further can be used as prevention, diagnosing and treating Biomarker in neoplastic process carries out related application.
Specific embodiment
Also the present invention further can be understood by embodiment, wherein the embodiment illustrates some preparations or user Method.It is to be appreciated, however, that these embodiments do not limit the present invention.The change of the invention of currently known or further exploitation Change is considered within the scope of the invention described herein and claimed below.
The correlation research of embodiment 1miRNA expression and Eag1 protein expression
Take the highly expressed human cervical carcinoma cell lines Hela of known Eag1 albumen and normal human blood sample.
1) RNA is extracted and MicroRNA is detected
The Trizol reagent of 3 times of volumes, ultrasonic Syrup-homogenizing instrument powder is added according to RNA in the extraction of Trizol method in cell or sample Broken tissue；Mutually separate:It is placed at room temperature for 15min, isometric chloroform is then added, shakes 50s, room temperature 15min, 14000rpm, 4 DEG C, it is centrifuged 15min；RNA precipitate：Water phase is transferred to the centrifuge tube of new 15ml, the anhydrous second of 1.5 times of water phase volumes is added Alcohol mixes well；RNA is enriched with QIAGEN miRNeasy kit：700ul sample is drawn every time into centrifugal column, 14000rpm is centrifuged 15s, discards filtrate in collecting pipe, repeat to cross column to sample standard deviation；Be added 700ul washing lotion 1,14000rpm from Heart 15s abandons filtrate in collecting pipe；500ul washing lotion 2 is added, 14000rpm is centrifuged 15s, abandons filtrate in collecting pipe；It adds 500ul washing lotion 2,14000rpm are centrifuged 2min, abandon filtrate in collecting pipe；Centrifugal column is put back in empty collecting pipe, 14000rpm 2min is centrifuged to dry centrifuge tube；Centrifuge tube is put into new 1.5ml pipe, be added 50ul nuclease-free water, 10,000rpm from Heart 1min；Liquid in pipe is refunded in centrifugal column, 14000rpm is centrifuged 1min, abandons centrifugal column；Measure concentration：Through 1% formaldehyde Denaturing Agarose Gel electrophoresis detection RNA integrality, and application Bio-Rad detection RNA concentration and purity, select electrophoretic band clear Clear (28S, 18S, 5S), A260/A280 are that 1.8-2.0 person is used for subsequent experimental.
Reverse transcription is carried out using microRNA Reverse Transcriptase kit (Applied Biosystems USA).Reaction system is 20ul, including：10×RT buffer 1.5ul,100mmol/L dNTPs 0.15ul,50U/ul MultiScribeTM Reverse Transcriptase 1.0ul, 20U/ul RNase inhibitor 0.19ul, 1umol/L reverse transcription specific primer 3ul, RNA template 1ul, DEPC water supply 20ul.Reaction condition：16 DEG C, 30min；42 DEG C, 30min；85 DEG C, 5sec；Product- 20 DEG C freeze it is spare.MiR-320 reverse transcriptase primer sequence such as SEQ ID NO：Shown in 2.Through sequencing confirmation reverse transcription product and SEQ ID NO：1 sequence is corresponding, ensure that sequence accuracy.
Taqman real-Time PCR：Using microRNA Real-Time RCR kit (Applied Biosystems USA) carry out pcr amplification reaction.Reaction system is 20ul, including：TaqMan GEx MASTER Mix 10ul, RT product 1ul, real time specific primer 1ul, distilled water supply 20ul.Reaction condition：95 DEG C, 10min；95℃ 15sec, 60 DEG C of 1min, totally 40 recycle.Specific PCR primers sequence such as SEQ ID NO：3 and 4.
The calculation method of microRNA expression：In the target gene situation identical as internal reference gene magnification efficiency Under, it using U6 as internal reference, measures cycle threshold (Ct value), calculates △ Ct value, △ Ct=miRNACt value-U6Ct value, with 2-△△CT Indicate the relative expression levels of miRNA.
2) in Hela cell and blood sample Eag1 albumen ELISA detection
Hela cell protein extracts：Cell protein is extracted according to conventional method in that art, ultrasound homogenate, centrifuging and taking Supernatant, BCA are quantitative.Then ELISA kit is prepared using method disclosed in Chinese patent CN201720900420, carries out Eag1 Protein Detection.
Concrete outcome see the table below：
That there is conspicuousnesses is poor for Eag1 protein content and miRNA content in normal sample it can be seen from upper table result It is different, and its expression quantity of miRNA provided by the invention is positively correlated with Eag1 albumen, can be used in clinic to tissue samples The assessment of Eag1 albumen.
Embodiment 2miRNA expression and the relationship of cervical carcinoma are investigated
The blood sample of 26 cervical cancer patients and 15 normal persons is collected in hospital, is carried out according to the method for embodiment 1 MicroRNA content detection, concrete outcome are as follows：
microRNA Cervical cancer patient Normal person
Expression quantity 1.49±0.42 0.36±0.13
It can be seen that the MicroRNA expression quantity exists obvious in cervical cancer patient and the blood sample of normal person Difference prompts microRNA of the invention to can be used as the molecular marked compound of clinical diagnosis of cervical cancer, and without carrying out biopsy It can carry out coherent detection.
The content of present invention merely illustrates claimed some specific embodiments, one of them or more skill Documented technical characteristic can be combined with arbitrary one or more technical solutions in art scheme, these are combined and obtain Technical solution also in the application protection scope, the technical solution just as obtained from these are combined is disclosed in the present invention It is specifically recorded in content the same.
Sequence table
<110>Jilin Engineering Normal College
<120>Specificity MicroRNA sequence relevant to potassium-channel Eag1 expression and its application
<130> 1
<141> 2018-06-13
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> RNA
<213> Homo sapiens
<400> 1
guauaguuca gccaggaauc ccu 23
<210> 2
<211> 44
<212> DNA
<213> Homo sapiens
<400> 2
ctcaactggt gtcgtggagt cggcaattca gttgagaggg attc 44
<210> 3
<211> 31
<212> DNA
<213> Homo sapiens
<400> 3
acactccagc tggggtatag ttcagccagg a 31
<210> 4
<211> 16
<212> DNA
<213> Homo sapiens
<400> 4
tggtgtcgtg gagtcg 16

Claims (7)

1. a kind of specificity microRNA, sequence such as SEQ ID NO relevant to potassium-channel Eag1 expression：Shown in 1 Nucleotide sequence or with its have at least 70% sequence identity, preferably have 80%, 85%, 90%, 93%, 95%, 97%, the nucleotide sequence of 98%, 99% sequence identity.
2. application of the microRNA sequence described in claim 1 in preparation detection Eag1 protein reagent box.
3. application of the microRNA sequence described in claim 1 in preparation lesion detection kit.
4. application according to claim 3, the tumour is selected from cervical carcinoma, oophoroma or lung cancer.
5. a kind of kit of microRNA sequence described in detection claim 1, the amplification containing the microRNA sequence Primer and the necessary reagent for extracting RNA.
6. kit according to claim 5, the sequence of the amplimer such as SEQ ID NO：Shown in 2-4.
7. kit according to claim 5, the kit also includes enzyme and reagent used in PCR reaction.
CN201810604285.8A 2018-06-13 2018-06-13 Specificity MicroRNA sequence relevant to potassium-channel Eag1 expression and its application Withdrawn CN108841825A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014082083A1 (en) * 2012-11-26 2014-05-30 Caris Science, Inc. Biomarker compositions and methods
CN206930677U (en) * 2017-07-24 2018-01-26 吉林工程技术师范学院 A kind of cervical carcinoma quick detection kit using Eag1 potassium-channel proteins as target

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014082083A1 (en) * 2012-11-26 2014-05-30 Caris Science, Inc. Biomarker compositions and methods
CN206930677U (en) * 2017-07-24 2018-01-26 吉林工程技术师范学院 A kind of cervical carcinoma quick detection kit using Eag1 potassium-channel proteins as target

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIANG LUO等: "Decreased miR-320 expression is associated with breast cancer progression, cell migration,and invasiveness via targeting Aquaporin 1", 《ACTA BIOCHIM BIOPHYS SIN》 *
龙青青等: "调控钾通道的非编码RNA在疾病中的研究进展", 《国际遗传学杂志》 *

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Application publication date: 20181120